The androgen receptor (AR) belongs to the superfamily of nuclear receptors that become active in gene transactivation in response to their ligands. Because the AR plays a central role in growth and survival of prostate cancer (PCa) cells, androgen deprivation therapy (ADT) has been a standard treatment for advanced/metastatic PCa. Unfortunately, a majority of tumors invariably relapse and become a disease called androgen-deprivation independent or castration-resistant prostate cancer (CRPC), from which most patients eventually die. Increasing evidence obtained from both clinical and experimental studies indicates that the AR remains active in the absence or presence of residual (castration) levels of androgens and plays a pivotal role in the development of CRPC. These findings have elevated the need to understand the molecular mechanisms by which the AR becomes promiscuously activated following ADT. Our preliminary data demonstrate that loss of the PTEN tumor suppressor gene increases AR activity in both the presence of low levels of androgens and their complete absence. This effect of PTEN is mediated primarily by its downstream nuclear factor FOXO1. Silencing of FOXO1 in PTEN-positive cells not only increases AR basal activity, but also sensitizes the AR to activation by very low concentrations of androgens and nonandrogenic factors such as interleukin-6 (IL-6). In contrast, forced expression of FOXO1 in PTEN-null PCa cells inhibits both androgen-dependent and -independent activation of the AR. Mechanistically, FOXO1 directly binds to AR's transcription activation unit 5 (TAU5) domain, a region known to interact with a number of functionally important transcription coactivators. The AR inhibitory function of FOXO1 requires its nuclear localization but not its transcriptional activity. Most importantly, expression of the transcription-deficient but AR-inhibitory mutant of FOXO1 inhibits growth of CRPC cells. STAT3 is important for IL-6-induced activation of the AR through its interaction with AR. We demonstrate that forced expression of the nuclear form of FOXO1 diminishes activation of the AR by IL-6 and the STAT3-AR interaction. Moreover, our in vitro studies show that the increase of AR activity with PTEN loss and FOXO1 inactivation is completely abolished by treatment of cells with small inference RNA of p300, an important coactivator of the AR. Conditional knockout of p300 in the mouse prostate shows that deletion of p300 inhibits AR activity in vivo. The unifying hypothesis of this application is that in prostatic cells with a functional PTEN, FOXO1 localizes primarily in the nucleus where it forms a protein complex with the AR that limits the association of the AR with coactivators and thereby prohibits aberrant AR activation. In PTEN-mutated PCa cells, however, highly activated Akt leads to phosphorylation and nuclear exclusion of FOXO1, which results in unopposed access of the AR to the coactivators such as p300 and STAT3 that promotes promiscuous AR activation in the presence of castration levels of androgens or nonandrogenic factors (e.g. IL-6) and castration-resistant disease progression. We propose three specific aims to test this hypothesis: (1) to define the precise mechanisms that mediate the inhibitory effect of the PTEN/FOXO1 signaling axis on androgen- independent activation of the AR and castration-resistant growth of PCa cells using xenograft models; (2) to determine the importance of p300 in androgen-independent and yet AR-dependent progression of prostate tumors in the PTEN-knockout model; and (3) to elucidate the molecular mechanism and biological importance of the PTEN/FOXO1 pathway in regulation of IL-6-induced androgen-independent activation of the AR in PCa cells. Information obtained from this study will not only shed new light on the molecular mechanism of aberrant AR activation in CRPC, but also define new molecules and pathways for targeted therapy of this fatal disease. PUBLIC HEALTH RELEVANCE: A majority of prostate cancers invariably relapse after androgen deprivation therapy and eventually become a castration-resistant disease, from which most patients eventually die. Increasing evidence suggests that the androgen receptor plays an essential role in the progression of castration-resistant prostate cancer due to its promiscuous activation. This project is designed to define the molecular mechanism by which the PTEN/FOXO1 signaling pathway regulates androgen receptor activity in response to low levels of androgens or nonandrogenic factors in cultured cells and in animals. Findings from this study will not only shed new light on the molecular mechanism of aberrant AR activation in castration-resistant prostate cancer, but also define new molecules and pathways for targeted therapy of this fatal disease.